Lecture: 1 Molecular Basis of Cancer

What Is Cancer? • Cancer is a group of diseases caused by the uncontrolled multiplication of abnormal cells in the body, a process called neoplasia. • Abnormal new tissues called neoplasms are formed. • Neoplasms usually form masses called tumors that may be benign (non cancerous) or malignant (cancerous). • Malignant or cancerous tumors grow rapidly,are invasive (to surrounding tissue) and metastatic (traveling via blood/lymph to invade distant tissues). • Cancers destroy healthy tissues causing loss of function and death. • Cancer is the 2nd major killer in populations of developed countries & the leading cause of death in children 3-15 (US). • Cancers are genetic disorders caused by accumulation of somatic mutations (gene & chromosome) in a person’s cells. • Inherited mutations give a predisposition for certain cancers.

Characteristics of Cancer Cells • Cancer cells are genetically altered via gene or chromosome mutations so: - lack normal controls over cell division or apoptosis. - may express inappropriate genes (e.g. for telomerase, enzyme that maintains length of DNA for continued division) - are genetically unstable due to loss of DNA repair mechanisms (so are more susceptible to radiation damage than normal cells). • Divide excessively (proliferate) & indefinitely producing neoplasms. • Live indefinitely (do not show apoptosis). • Lose the normal attachment to other cells so become metastatic (travelling via blood/lymph to invade distant sites). • Secrete signals for angiogenesis (growth of blood vessels into tumor).

Cancer Cells are Undifferentiated & Malignant undifferentiated to varying degrees (even anaplastic, like stem cells) so divide & do not perform the normal function of mature cells. • The less differentiated the cancer cell the more malignant the cancer (the more rapidly growing is the tumor).

What Causes Cancer? Inherited mutations in genes that affect cell cycle, DNA repair, or apoptosis: these mutations give a genetic predisposition for cancer. Somatic mutations to these same genes caused by: • Exposure to risk factors - environmental mutagens (carcinogenic chemicals, radiation) - hormones - weakening of immune system (as in AIDS). • Oncogenic (tumor) Virus infections - Epstein Barr virus (causes Burkitt lymphoma) - Human Papilloma Virus (causes cervical cancer). Tumor viruses transform human cells into cancer cells by: •Introducing viral cancer - causing oncogenes into host cell DNA •Causing Translocation and overexpression of host protooncogenes.

Normal cell cycle is controlled by signal transduction: Growth factors bind to surface receptors on the cell; transmembrane proteins relay signals into the cell. Two types of growth factors: Growth factors stimulate cell division. Growth-inhibiting factors inhibit cell division. Healthy cells divide only when growth factor and growth-inhibiting factor balance favors cell division. Cancer cells divide without constraint (e.g., mutations in growth and growth-inhibiting factor genes).

Regulation of cell division by signal transduction.

The Cell Cycle Oncogenes G2 (cell growth) M (mitosis) S (synthesis) DNA repair genes REPAIRS AHEAD G1 G0 (resting) Tumor suppressor genes

Cell cycle CDK p53 is known as the ‘ guardian of the genome ‘

CONTROL of CELL CYCLE Differentiated The Cell has 3 major Cells enter G0 Cells enter “checkpoints” that are non dividing G1 sensitive to signals, the checkpoint G0 phase- G1, the G2 and the M checkpoints. • If a cell does not G1 pass G1 checkpoint Control it enters a non- system S dividing GO Phase. • Most somatic cells G2 are in GO. M • Some cells in GO (e.g. liver cells) can reenter cell cycle if M checkpoint needed. G2 checkpoint In cancer cells genes that control cell cycle have mutated so cells divide excessively producing neoplasms.

Cancer and genes: Three classes of genes are frequently mutated in cancer: Proto-oncogenes ( oncogenes) Tumor suppressor genes Mutator genes

Proto-oncogenes  oncogenes: Proto-oncgenes are genes that possess normal gene products and stimulate normal cell development. Oncogenes Oncogenes arise from mutant proto-oncogenes. Oncogenes are more active than normal or active at inappropriate times and stimulate unregulated cell proliferation. Some tumor viruses that infect cells possess oncogenes: RNA tumor viruses = possess viral oncogenes (derived form cellular proto-oncogenes) capable of transforming cells to a cancerous state. DNA tumor viruses = another class of tumor viruses; do not carry oncogenes, but induce cancer by activity of viral gene products on the cell (no transformation per se).

Types & effects of different types of mutations: Point mutations: occur in protein coding or controlling sequences. Deletion: frameshifts may lead to defective proteins. Gene amplification: random over-replication of small segments of DNA results in extra copies (up-regulates cell growth). Mutator genes: Mutator gene increases spontaneous mutation rate of other genes. Mutator gene products are involved in DNA replication and repair; mutations make the cell error prone.

Proteins Control Cell Division The cell cycle is controlled by proteins from inside & outside the cell. • Intracellular Cyclins and Cyclin Dependent Kinases (CDKs) control the checkpoints. • Hormones or extracellular proteins from other cells (called Growth Factors) signal target cell to divide. - Hormones (e.g. Growth Hormone) or Growth Factors bind to receptor proteins of target cell membrane. - This triggers a molecular signaling pathway. - A series of linked proteins activate Cyclin- CDKs which Allows Cells to Pass Cell Cycle Checkpoints & divide.

How Growth Factors Trigger Cell Division or Hormone Plasma membrane Relay •G1 checkpoint prevents Proteins open damaged DNA from replicating checkpoints •Checkpoint controlled by Receptor Cyclin - CDK protein Signal Transduction Control Pathway G1 S system M G2

Tumor Suppressor Proteins Inhibit Cell Division & Prevent Cancer Tumor suppressor proteins are proteins that bind to checkpoint proteins to stop the cell cycle & prevent cell division if DNA is damaged. • Tumor suppressor proteins stop division of mutated cells until mistakes in DNA are repaired by enzymes. • TS proteins keep most mutations from being passed on to daughter cells & developing into cancer. • If the genes for TS proteins mutate the brake on cell division is removed cancers may result. • Two important TS proteins are the p53 protein & the RB protein.

The p53 Tumor Suppressor Protein The p53 tumor suppressor protein is activated when DNA is damaged. The p53 gene is called the “guardian angel of the genome” P53 protein activates Internal genes for signalling pathway proteins that •Prevent cell entering S phase •Repair DNA •Cause DNA repair apoptosis (if Apoptosis DNA is Cell cannot enter S phase irreparable)

Gene Mutations That Cause Cancer Mutations in 4 types of genes cause Cancer • Proto - oncogenes: genes that code for normal proteins used in cell division –Growth factors –Membrane Receptors for Growth Factors –Signaling Proteins (e.g. ras proto- oncogene mutates in 30% of cancers). • Tumor Suppressor genes: gene that code for proteins that help prevent uncontrolled cell division by blocking key steps (e.g. DNA replication). - Retinoblastoma susceptibilty (RB) gene - p53 gene mutates in >50% of cancers. • DNA Repair genes • Genes for Apoptosis

How Carcinogens Cause Cancer Inactivation of DNA Repair Genes Oncogenes of Genes for Tumor Apoptosis Suppressor Genes

Oncogenes Are Mutated Proto-oncogenes A cell can acquire a cancer - causing oncogene from •A virus •A mutation in a proto-oncogene. Oncogenes still code for the proteins needed for cell division but they cause cancer by producing – Too much of the protein – An abnormally active protein, e.g. protein that activates division by itself – Protein that is made when it is not needed – Protein that should be made by a different (i.e. dividing) cell.

Cancer causing Mutations •Proto-oncogenes form active oncogenes by - being misplaced (e.g. by translocation) to a site where the gene is continually expressed resulting in overproduction of a protein that stimulates cell division (e.g. in Chronic Myeloid Leukemia) - By mutating to a form that is over expressed. •Cancer causing Mutations in Tumor Suppressor genes inactivate the genes so normal protein product is not formed. Mutated oncogene Tumor Suppressor gene neoplasm

Over Stimulation Hyperactive of Cell relay protein Division by Growth Stimulation Membrane factor of Cell Receptor Hyperactive Division by relay protein Oncogene Normal product (product of of ras gene ras oncogene) Relay issues signals proteins on its own. Transcription factor (activated) DNA Transcription Protein that stimulates Translation cell division

Cancer from Mutation of Tumor Supressor Gene Tumor-suppressor gene Mutated tumor-suppressor gene Cancer Normal from Protein Mutation Defective, prevents of Tumor nonfunctioning cell Supressor protein division if DNA is Gene Protein damaged absent (cell division Cell division not blocked) allowed if DNA repaired Mutations accumulate in cancer cells

Multiple Genetic Changes Cause Cancer Cancers result from a series of genetic changes in a cell lineage • Inherited (germline) cancers begin with an inherited cancer susceptibility mutation in every cell that is passed on to offspring. • Inherited cancers may follow a dominant pattern, e.g. Inherited Retinoblastoma caused by a mutation in the Rb tumor supressor gene increases cancer risk 10,000 x. • However, Inherited cancers need at least one more somatic mutation for cancer to develop (“2 hit hypothesis for cancer causation”) . • Sporadic cancers are caused solely by somatic mutations occurring in certain body cells so are not passed on to offspring. • Accumulation of somatic mutations in a cell over time eventually leads to uncontrolled cell division and cancer. • Therefore sporadic cancers tend to appear much later in life than inherited cancers.

Accumulation of Mutations Cause Cancer • inheritance of a germ cell mutation acts as a risk factor for cancers by reducing the number of somatic mutations required to cause cancer. • Early mutations show up in all subsequent stages of a cancer. Normal 1 4 2 3 Chromosomes mutation mutations mutations mutations Normal cell Malignant cell

Oncogenes Normal genes (regulate cell growth) 1st mutation (leads to accelerated cell division) 1 mutation sufficient for role in cancer development

Tumor Suppressor Genes Normal genes (prevent cancer) 1st mutation (susceptible carrier) 2nd mutation or loss (leads to cancer)

The Two-Hit Hypothesis First hit First hit in germline of child Second hit (tumor)

Pathogenesis of Colon Cancer Colon Cancer is usually Sporadic & develops in a series of steps caused by a series of somatic cell mutations Loss of Removal of polyps tumor- prevents cancer suppressor Colon wall gene (e.g. APC) Small benign Normal colon growth (polyp) epithelial cells = adenoma APC: adenomatous polypodsis coli

Pathogenesis of Colon Cancer -2 Activation of Loss of Campbell, & Reece ras oncogene tumor- Biology fig. 19.13 suppressor gene p53 Loss of Additional tumor- mutations suppressor Malignant tumor Larger benign Small benign gene (adenocarcinoma) growth (adenoma) growth (polyp) Environmental Risk factors for Colon Cancer • Low fiber diet • Smoked meats (contain heterocyclic aromatic amines that are converted to mutagens in the liver) • Low intake of fruits & vegetables (antioxidants) • Low intake of cruciferous vegetables (a chemical in brocolli, brussels sprouts, cabbage activates enzymes that block formation of mutagens)

Genetic Abnormalities Associated With Hematologic Malignancies A- Point Mutaion Mutaions within the RAS oncogenes or P53 tumor- suppressor gene are common in many haempoietic malignancies. The point mutation may involve several base pairs. In 35% of cases of AML the nucleophosmin gene shows an insertion of 4 base pairs. B- Translocation Includes two main mechanisms: 1- Fusion of parts of two genes to generate a chimeric fusion gene that codes a novel fusion protein. Ex: BCR- ABL in t(9; 22) in chronic myeloid leukaemia. 2- Overexpression of a normal cellular gene. Ex: overexpression of BCL-2 in the t(14; 18) translocation of follicular lymphoma or MYC gene in Burkitt,s lymphoma.

D- Duplication or amplification E- Epigenetic alterations C- Deletions May involve a small part of a chromosome, the short or long arm or the entire chromosome. Losses most commonly affect chromosomes 5, 6, 7, 11, 20 and Y. The critical event is probably loss of a tumor suppressor gene. D- Duplication or amplification Gains are common in chromosomes 8, 12, 19, 21 and Y. It is not a common feature in haematologic malignancy but has been described involving the MLL gene. E- Epigenetic alterations Means alterations in the mechanism by which genes are transcribed and are stably inherited with each cell division so they are passed on as the malignant cell divides. The most important mechanisms are methylation of cytosine residues in DNA and enzymatic alterations such as acetylation or methylation of the histone protein that package DNA.

Prostate Cancer • Prostate Cancer is the most common cancer among men (esp. >65 yrs, African - Americans) & 2nd in cancer deaths in men. • Risk factors include increasing age, race, family history, fat diet, male hormones over many years. • Adenocarcinoma occurs in periphery of prostate gland. • Metastasises to lungs, bones (bone pain often first symptom as early stage of primary tumor may be asymptomatic). • Tumors are graded from 1 (well differentiated cells) to 5 (least differentiated cells, high malignancy). • Manifestations (some similar to Benign Prostatic Hyperplasia): - changes in voiding pattern, dysuria, hematuria, – from metastasis low back pain from bone, wt loss, anemia, & shortness of breath. • Screening (important for early asymptomatic cancers) includes: - digital rectal exam (palpation of prostate by DRE detects nodular lump) - transrectal ultrasonography (measures prostate vol: more sensitive than DRE) - PSA blood test

PSA Test for Prostate Cancer • Tumor cells express abnormal genes so form abnormal proteins (antigens) so can act as tumor markers. • Prostate Specific Antigen (PSA) a glycoprotein released by prostate gland into the blood identified as a marker of prostate cancer in 1980. • PSA is highly specific to prostate gland but not specific to prostate cancer. Elevated blood PSA can also occur with non cancerous conditions (i.e. false positives occur with prostatitis or with benign prostatic hyperplasia - BPH) • The need to treat stage 1 cancers detected by PSA test is controversial: stage 1 tumors are asymptomatic, not detected by digital exam & present in 80% of men over 80 yrs. • Treatment of Prostate Cancer includes surgery, radiation & hormonal manipulation (e.g. androgen inhibitors) if expected survival >10yrs, otherwise “watchful waiting” is preferred in elderly patients. • PSA test is used to assess treatment (correlates with prostate size & cancer stage).

Etiology of Breast Cancer Breast cancer is most common cancer in women & 2nd most common in cancer deaths in women (after lung cancer). Risk Factors for Breast Cancer • Prolonged exposures to estrogens (early menarche & late menopause). Breast cancers that are estrogen receptor +ve are treated with drugs (e.g. tamoxifen) that bind to these receptors. • Late Childbearing (having first child after age 30) • Breasts with a high proportion of lobular (milk producing) and ductal tissue density. • Not breast feeding babies increases post menopausal BC. • Exposure to radiation. • High alcohol consumption. • Family History of BC & Genetic Predisposition in 5-20% of cases (inheriting mutated breast cancer susceptibility genes, BRCA-1 or BRCA-2).

Genetics of Breast Cancer • 5 - 20% of breast cancers are Familial . • Most involve mutations in 2 Tumor Suppressor genes involved in DNA repair so are used as genetic markers. • Both genes also increase the risk of ovarian cancer. - Breast Cancer Susceptibility Gene 1 (BRCA1) on chromosome 17 - Breast Cancer Susceptibility Gene 2 (BRCA2) on chromosome 13. • Mutated HER-2/neu Gene (Human Epidermal Growth Factor Receptor 2 gene is an Oncogene for a protein that stimulates cell division & occurs in 25- 30% of Breast Cancers. • Her-2/neu breast cancers strike early in adulthood & spread quickly. • Herceptin is a monoclonal antibody based drug that binds to Her-2/neu receptors & blocks cell division in tumors.

Pathogenesis of Breast Cancer Tumor (usually adenocarcinoma in milk ducts) Campbell, & Reece, Biology, fig. 12.19 Glandular tissue Note: lumpectomy a possible treatment in early stages A tumor grows from a Cancer cells invade single cancer cell. neighboring tissue.

Pathogenesis of Breast Cancer -2 Lymph vessel Blood vessel Cancer cell Metastatic tumor • Cancer cells spread via Small percentage lymph & blood to other of metastisised cancer parts of the body. cells may survive and • “Sentinel lymph node ” biopsy determines if establish a new tumor in a cancer has spread & if another part of the body. further lymph node removal required.

Diagnostic methods used to study malignant cells 1- Karyotype analysis: It is a direct morphological analysis of chromosomes from tumor cells under the microscope. 2- Fluorescent in situ hybridization analysis FISH analysis involves the use of fluorescent- labelled genetic probes which hybridize to specific parts of the genome. This can detect extra copies of genetic material or reveal chromosomal translocation. 3- Southern blot analysis: It involves extraction of DNA from leukaemic cells followed by restriction enzyme digestion, gel electrophoresis and transfer by blotting to a suitable membrane. The DNA is then hybridized to a probe complementary to the gene of interest. 4- Polymerase chain reaction: Can be performed on blood or bone marrow for a number of specific translocations such as t(9; 22) and t(15; 17). It is very sensitive and can detect one abnormal cell in one million normal cells. It is of great value to diagnose minimal residual disease.

5- DNA microarray:Allows rapid and comprehensive analysis of cellular transcription by hybridizing labelled cellular mRNA to DNA probes which are immobilized on a solid support. This approach can rapidly determine mRNA expression from a large number of genes and may be used to determine the mRNA expression pattern of different leukaemia or lymphoma subtypes. 6- Flow cytometry: Normal cells each have a characteristic profile but malignant cells often express an aberrant phenotype that can be useful in allowing their detection. 7- Immunohistochemistry: Antibodies can be used to stain tissue sections with fluorescent markers. Value of using these methods: a- Initial diagnosis. b- Establishing treatment protocol. c- Monitoring response to therapy.

Cytogenetics • Cytogenetics is the original cancer genetic test used to identify abnormal mutated chromosomes by karyotype analysis. • Cytogenetics identified the Philadelphia chromosome resulting from a translocation error in chromosome 22 forming an oncogene for chronic myelogenous leukemia (CML) in 1960. • Karyotype Analysis is done by culturing tumor cells, arresting them in metaphase & spreading chromosomes via use of hypotonic solutions. • Chromosomes are stained and interpreted by a cytogeneticist. • Process may take weeks (as many tumors do not grow in vitro).

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